Cryo-EM has emerged as an indispensible tool to visualize biological structures at high resolution. Despite the growing enthusiasm for this field, cryo-EM is a relatively inaccessible method for several reasons: first, there are inadequate training materials for cryo-EM novices; second, there is a growing expertise gap between experienced practitioners and newcomers; and third, few institutions have the means to procure and maintain state-of-the-art instrumentation. To address these limitations, we propose to create a self-paced, interactive online course using a media-rich and user-friendly approach that targets cryo-EM newcomers. Our curriculum aims to bridge the expertise gap by providing practical guidance on steps along the standard single particle cryo-EM project workflow. Our centralized ?CryoEM-101? website will bring cryo-EM principles and protocols to life using video, 3D animation, and interactive simulations. The curriculum will cover pertinent topics divided into five chapters: sample purification, grid preparation and optimization, grid screening and evaluation, data collection, and data processing. Each chapter consists of an overview summary and a list of learning objectives, followed by individual, digestible modules that focus on a specific concept along the workflow. Importantly, our curriculum will teach users how to maximize their efforts using modest instrumentation. Overall, the goal of this proposal is to guide newcomers to a point along their training where they can independently prepare and optimize their cryo-EM specimens in preparation for high-quality data collection. We will have an Advisory Board that consists of cryo-EM experts to evaluate our curriculum as it is developed. Local cryo-EM trainees will be recruited to test the training material and feedback will be sought through discussion group meetings and voluntary surveys. Upon launch of the Cryo-EM 101 website, the course will be shared broadly through announcements at meetings, social media, and the design of a massive open online course (MOOC).